Up until FY 2008, the ID09B time-resolved X-ray beamline at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France was the only facility in the world capable of determining time-resolved macromolecular structures with 150-ps time resolution and <2-Angstrom spatial resolution. The Anfinrud group was instrumental in helping develop that capability at the ESRF. Unfortunately, the ESRF operated in a mode that was optimized for time-resolved Laue crystallography studies only 14 days out of each year, and we had access to only a portion of this limited amount of beam time. To expand the amount of beam time available for our studies, we partnered with the Advanced Photon Source (APS) in Argonne, IL and BioCARS to develop picosecond time-resolved X-ray capabilities on the ID14B beamline. In FY2005, Dr. Marvin Gershengorn, then Director of Intramural Research at NIDDK, committed >$1M to procure the capital equipment needed for this effort. Our vision was to achieve picosecond time-resolved X-ray capabilities comparable to that realized at the ESRF when the APS is operated in 24-bunch mode, a common operating mode used 132 days per year. This goal required that we isolate a single bunch of X-rays from a train of pulses separated by only 153 ns, a feat that we first achieved in July 2007 using a high-speed chopper whose rotor was fabricated according to our custom specifications. To maximize the number of photons delivered to the sample in a single X-ray pulse, we replaced the existing U33 undulator (33-mm magnetic period) with two newly designed U23 and U27 undulators, making BioCARS the first APS beamline to operate with two inline undulators. NIDDK funded this effort, with the APS supplying the labor to design and refurbish two undulators according to our performance specifications. When the gaps of these undulators are tuned to generate 12-keV X-ray photons, the X-ray fluence is comparable to that generated at the ESRF during their 4-bunch mode. When the APS operates in their exotic hybrid mode, which is scheduled approximately 31 days per year, the X-ray fluence is a factor of 4 higher than that available with the ESRF 4-bunch mode. These achievements increased by more than an order of magnitude the amount of beamtime available worldwide to pursue 150 ps time-resolved X-ray science. The infrastructure needed to pursue picosecond time-resolved X-ray studies goes far beyond delivering single X-ray pulses to the experimental hutch. We also installed a $500K picosecond laser system in a laser hutch located near the X-ray hutch, as well as an array of laser diagnostics that aid optimization of the laser performance. We also developed a Field-Programmable-Gate-Array (FPGA) based timing system that synchronizes all time-critical components to the X-ray pulses. For example, the FPGA drives the heat-load chopper, the high-speed chopper, the picosecond laser system, a millisecond shutter, and various other motion controls that must be synchronized with the x-ray pulse arrival time. Importantly, we can set the time delay between X-ray and laser pulses from picoseconds to seconds with a precision of 10 ps. We also developed the diffractometer used to acquire time-resolved X-ray diffraction images. This effort included the design and fabrication of a millisecond shutter, a motorized support for the high-speed X-ray chopper, a support for motorized X-ray slits, detectors for non-invasively monitoring the laser and X-ray pulse energy and relative time delay, a motorized stage for the X-ray detector, supports for a collimator pipe and X-ray beam stop, beam conditioning optics that tailor the laser pulses in both space and time, beam delivery optics that focus the laser pulses onto the sample, motorized controls to center the focused laser pulse on the sample, and motorized controls to center the collimator pipe on the X-ray beam. Finally, we continue to refine the software developed to control the beamline. This software package, called LaueCollect, is written in the Python programming language, and is generalized for both time-resolved Laue crystallography and time-resolved SAXS/WAXS studies. For these experiments, it is crucial to achieve long-term stability of the laser and x-ray beams, Because of the large distance between the laser system and the sample (> 30 m), thermal drift in the laser hutch, the hall of the synchrotron ring, and the X-ray hutch caused the laser beam position to drift from its set point. We traced much of the long-term beam pointing problems to thermal instabilities, which affected the alignment of the beam conditioning optics. To mitigate this problem, we designed in Oct. 2012 a new support structure that secures the beam conditioning optics breadboard to the same optical table that supports the x-ray diffractometer components. This change markedly enhanced the stability of the laser beam focus, and improved the quality of data acquired since this change was made. In FY2012, the NIH provided funding to acquire a new, $1.5M high-speed, large area Rayonix x-ray detector, which was originally slated for delivery in Jan 2013. This detector is capable of recording 10 x-ray images per second, which is more than 25 times faster than the capabilities of the current mar165 CCD. Because our samples cannot tolerate repeated excitation at 10 Hz, at least not at the same spot, it is crucial to move the sample after each pump-probe measurement, which requires a motion control system capable of translating the sample to a new position with 1-2 um precision in well under 100 ms. The existing Huber Kappa diffractometer is outfitted with DC motors whose response is far too slow to keep up with the new detector. Therefore, we developed an alternative: a compact, high-speed diffractometer capable of repositioning the sample with 1g acceleration in all three axes with 1-2 um precision. This goal required that we keep the diffractometer small and light, which was achieved with three Parker MX-80 linear motor translation stages in an XYZ configuration. These stages are controlled by an Aerotech Ensemble multi-axis motion control system that also controls rotation of the sample about the phi axis. The mechanical system was designed and constructed in the winter of 2013, with the custom parts needed machined by Bernie Howder. This new instrument has been successfully tested and employed in time-resolved experiments conducted on the BioCARS beamline since Jun 2013. Unfortunately, the high-speed detector delivery date has slipped to Jan 2014, so we have not yet been able to take full advantage of this new capability. In addition to our instrument development efforts on the BioCARS beamline at the APS, we are developing the infrastructure required to pursue time-resolved X-ray studies at the Linear Coherent Light Source (LCLS) at Stanford with sub-ps time resolution. The LCLS is the worlds first free-electron X-ray laser, and is capable of generating extremely intense X-ray pulses with less than 100 fs duration. By exporting capabilities developed at the APS to the LCLS, we will extend the time resolution limits of our time-resolved X-ray studies by nearly 3 orders of magnitude. The information gained from such studies will help unravel the mysteries into how proteins function at the molecular level.